Compositions for treating epithelial and retinal tissue diseases

ABSTRACT

The present invention relates to mononucleoside phosphate compounds that have the benefits of a dinucleotide pharmaceutical. These mononucleoside phosphates can be made from a mononucleotide that has been modified by attaching a degradation resistant substituent on the terminal phosphate of a polyphosphate mononucleotide. By attaching this degradation resistant substituent, the stability from degradation matches or exceeds those of certain dinucleotides.

This application is a continuation-in-part of U.S. application Ser. No.09/643,138, filed Aug. 21, 2000.

TECHNICAL FIELD

The present invention relates to compounds and the methods of using suchcompounds in the diagnosis, prevention or treatment of epithelial andretinal tissue diseases or conditions of humans and other mammals. Suchdiseases or conditions requiring diagnosis, prevention, or treatment ofepithelial tissue diseases include respiratory diseases, eye diseases,vaginal and/or cervical dryness, gastrointestinal tract diseases, andinflammatory and allergic diseases.

BACKGROUND OF THE INVENTION

Epithelial tissues comprise a layer or layers of cells that cover freeand enclosed surfaces throughout the body, including cutaneous, mucous,lumenal, serous, and glandular spaces. All epithelial layers contain twospecialized domains: an apical domain that faces the mucosal (orlumenal) space and a basolateral membrane that faces the serosal (orablumenal) space. Thus an important function of all epithelia is toprovide an appropriate barrier function to separate and to control manyphysiological processes between these two spaces. In the lung, forexample, the airways epithelia serve many functions, including providinga barrier between the lung mucosa and blood supply, to coordinate thehydration of the airways, to regulate blood-borne immune responses inthe airway mucosa, and to clear the airways of toxins and pathogens.Epithelial cells are ubiquitous throughout the body, and are found inthe entire respiratory and digestive tract, reproductive system andsensory organs (eye, ear, nose and skin). Epithelial cells have evolvedto serve many homeostatic functions that are specific to their locationthroughout the body. One such specific function is found in themucociliary clearance (MCC) system. Mucous secretions are normallyremoved via the MCC. MCC relies on the integrated action of threecomponents: 1) mucus secretion by goblet cells and submucosal glands; 2)the movement of cilia on epithelial cells which propels the mucus acrossthe luminal surface; and 3) ion transport into and out of luminalepithelial cells which concomitantly controls the flow of water into themucus. It is now known that nucleoside phosphates such as uridine5′-triphosphate (UTP) modulate all of the components of the MCC system.First, UTP has been shown to increase both the rate and total amount ofmucin secretion by goblet cells in vitro (M. Lethem, et al., Am J.Respir. Cell Mol. Biol. 9, 315–22 (1993)). Second, UTP has been shown toincrease cilia beat frequency in human airway epithelial cells in vitro(D. Drutz, et al., Drug Dev. Res. 37(3), 185 (1996)). And third, UTP hasbeen shown to increase Cl⁻ secretion, and hence, water secretion fromairway epithelial cells in vitro (S. Mason, et al., Br. J. Pharmacol.103, 1649–56 (1991)). In addition, it is thought that the release ofsurfactant from Type II alveolar cells in response to UTP (Gobran, Am.J. Physiol. 267, L625–L633 (1994)) contributes to optimal functioning ofthe lungs and may assist in maximizing MCC. UTP has been shown toincrease intracellular Ca⁺⁺ due to stimulation of phospholipase C by theP2Y₂ receptor (H. Brown, et al., Mol. Pharmocol. 40, 648–55 (1991)).

The retinal pigment epithelium (RPE) lies in the back of the vertebrateeye and forms a barrier that separates the retina from the choroidalblood supply. Although anatomically an epithelial tissue, the RPE alsofunctions in a glial-like capacity in maintaining homeostatic retinalfunction. For example, a critical function of the RPE is to maintain andregulate the hydration of the subretinal space, the extracellular volumethat exists between the retina and the RPE. (Mannor, pp. 3–12, in TheRetinal Pigment Epithelium, Eds. M. F. Marmor and T. J. Wolfensberger,Oxford University Press, New York, (1998)) This function is achieved bythe regulated transport of fluid, ions, and metabolites between thesubretinal space and the choroidal blood supply. (Marmor, pp. 420–438,in The Retinal Pigment Epithelium, Eds. M. F. Marmor and T. J.Wolfensberger, Oxford University Press, New York, (1998); Pederson, pp.1955–1968, in Retina, Ed. S. J. Ryan, Mosby, St. Louis, (1994)). Likeall epithelia, the RPE contains two functionally and anatomicallydistinct membranes: an apical membrane that faces the retina, and abasolateral membrane that faces the choroidal blood supply. In thenormal retina, fluid is absorbed across the RPE in the direction of thesubretinal space to the choroid. This active absorption of fluid by theRPE, often referred to as the “RPE pump,” plays a critical role inmaintaining proper attachment of photoreceptors to the apical membraneof the RPE by pumping fluid out of the retinal spaces. (Marmor, pp.1931–1954, in Retina, Ed. S. J. Ryan, Mosby, St. Louis, (1994); Hughes,et al., pp. xvii, 745, in The Retinal Pigment Epithelium, Eds. M. F.Marmor and T. J. Wolfensberger, Oxford University Press, New York,(1998)).

Glaucoma is a disease complex characterized primarily by an increase inintraocular pressure. Sufficiently high and persistent intraocularpressure may result in damage to the optic disc at the juncture of theoptic nerve and retina, resulting in irreversible blindness. There arethree types of glaucoma: primary, secondary, and congenital. Primaryglaucoma is subdivided into narrow angle (acute congestive) andwide-angle (chronic simple) types, depending on the configuration of theangle of the anterior chamber where re-absorption of the aqueous humoroccurs. Effects on the volumes of the various intraocular vascular beds,such as those of the iris and ciliary body and on the rate of secretionof the aqueous humor into the posterior chamber may contributesecondarily to the lowering of the pressure or, conversely, may producea rise in pressure preceding the fall. In narrow angle glaucoma, theaqueous outflow is enhanced by freeing of the entrance to the trabecularspace at the canal of Schlemm from blockade by the iris, as a result ofthe drug-induced contraction of the sphincter muscle of the iris.(Taylor, pp. 123–125, in The Pharmacological Basis of Therapeutics,7^(th)Ed, Eds., A. G. Gilman, L. S. Goodman, T. W. Rall, and F. Murad,MacMillan Publishing Company, New York, (1985))

In wide-angle, or chronic simple, glaucoma, the entry to the trabeculacis not physically obstructed; the trabeculae, a meshwork of pores ofsmall diameter, lose their patency. Contraction of the sphincter muscleof the iris and the ciliary muscle enhances tone and alignment of thetrabecular network to improve re-absorption and outflow of aqueous humorthrough the network to the canal of Schlemm (Watson, Br. J. Opthalmol.56: 145–318 (1972); Schwartz, N Engi. J. Med., 290: 182–186 (1978);Kaufman, et al., Handbook of Experimental Pharmacology 69: 149–192(1984)).

Human joints are lubricated by fluid secreted from synovial membranes,which line internal, non-articular joint surfaces. The lubricatingproperties of synovial fluid have been attributed to a surfactantconsisting of surface active phospholipid (SAPL), the mucinousglycoprotein lubricin, hyaluronic acid (hyaluronan), and water.Hyaluronan is a critical constituent component of normal synovial fluidand an important contributor to joint homeostasis. Hyaluronan impartsanti-inflammatory and antinociceptive properties to normal synovialfluid and contributes to joint lubrication, buffering load transmissionacross articular surfaces and providing a continually replenished sourceof hyaluronan to articular tissues. Joint lubrication is compromised inosteoarthritis (OA).

Studies suggest that activation of P2Y receptors by extracellularnucleotides elicit responses from inflammatory cells (such as mastcells, eosinophil, leukocytes, neutrophils) consistent with apro-inflammatory effect. Extracellular nucleotide-induced stimulation ofleukocytes and subsequent adhesion to endothelium has been shown to playan important role in inflammatory diseases. Extracellular nucleotidesstimulate P2Y receptor on human polymorphonuclear neutrophils (PMN) withthe pharmacological profile of the P2Y₂ receptor.

Allergy is a state of hypersensitivity caused by exposure to a specificantigen (allergen) resulting in harmful immunologic reactions onsubsequent exposures. The first encounter with an allergen sensitizesthe body via the lymphocytes, resulting in IgE coating of mast cells andbasophils. Subsequent exposure results in the development of the “earlyphase” of the allergic reaction and occurs within seconds or minutes ofexposure to an allergen. The early phase is also known as the immediatehypersensitivity reaction. In the allergic reaction, hypersensitivity isa condition in a previously exposed person, in which tissue inflammationis caused by an immune reaction upon re-exposure to an allergensensitizer. In half of occurrences, the allergic reaction develops intoa “late phase,” which occurs about 4 to 6 hours after the exposure. Inthe late phase reaction, tissues become red and swollen due to thecollection of eosinophils, neutrophils, lymphocytes, and other cells.Preferably the nucleotide receptor is a P2Y purinergic receptor, such asthe P2Y₂ receptor. Activation of such receptors by P2Y agonists triggerthe elevation of intracellular calcium levels and activation ofsignaling pathways leading to prevention and/or reversal of the symptomsand manifestations of early and late phases of allergic reactions andinflammatory diseases. Previous work has demonstrated the presence ofP2Y receptors in glial and neuronal cells of the mature nervous system(Abbracchio and Bumstock, Jpn J. Pharmacol, 78:113–45, 1998). P2Yreceptors belong to a class of G-protein coupled receptors (GPCR) thatactivate a variety of intracellular signaling pathways. Althoughfeatures of P2Y receptor signaling in many cell types are well known,the physiological roles of P2Y receptors in the nervous system are notwell-characterized. In central, peripheral and sensory nervous systems,P2Y receptor activation profoundly affect glia, a cell type that playsimportant roles in nervous system development, function, and survival.Previous work has suggested a role for P2Y receptors inneurotransmission, neuronal-to-glial cell-cell signaling, alterations ofgene expression, neuritogenesis, and interactions with growth factors inan additive or synergistic manner (Abbracchio and Burnstock, Jpn JPharmacol, 78:113–45, 1998).

There is an unmet medical need for new therapeutic nucleotides that havegood storage stability and/or in vivo stability that can be used for thetreatment of epithelial and retinal diseases with minimal side effects.Nucleotides, defined here as a nucleoside base with one or morephosphate groups attached at the furanosyl primary hydroxyl group, canact via receptors (e.g. P2Y), and ion channels (e.g. P2X). Thetherapeutic utility of nucleotides arises from their actions as eitheragonists or antagonists of receptor (P2) function. Two classes oftherapeutic nucleotides have emerged recently—mononucleotides (e.g.nucleoside tri- and diphosphates) and dinucleotides (dinucleosidepolyphosphates). Mononucleotides, such as uridine triphosphate andadenosine triphosphate (UTP and ATP) are potent ligands of P2 receptors(see U.S. Pat. Nos. 5,292,498 and 5,628,984). However thesemononucleotides have poor chemical and metabolic stability making themless attractive as drug candidates due to required refrigeration andshort in vivo half-life. Dinucleotides, such as diuridine tetraphosphateand diadenosine traphosphate (Up₄U and Ap₄A), show an improvement inchemical and metabolic stability while retaining activity at various P2receptors (see U.S. Pat. Nos. 5,635,160; 5,837,861; 5,900,407;6,319,908; and 6,323,187).

Despite the therapeutic improvements made by the use of dinucleotidesand their in vivo and storage stability, the difficulty and expense oftheir synthesis requires further improvement for use in the treatment ofepithelial and retinal diseases with minimal side effects.

SUMMARY OF THE INVENTION

The present invention comprises compounds of mononucleoside phosphatesof the general Formula I, or pharmaceutically acceptable salts thereof:

wherein;A has a molecular weight of no more than about 1000 and is OR₁, SR₁,NR₁R₂, or CR₁R₂R₃ such that R₁, R₂, and R₃ are each independentlyhydrogen, alkyl, cycloalkyl, aryl, arylalkyl, phosphonate, oracylthioalkyl, with or without substituents or heteroatoms; or takentogether to form a cycloalkyl or aryl ring, with or without substituentsor heteroatoms, with the exception of OR₁ and SR₁ not being OH or SH; ora natural or non-natural amino acid, peptide, polypeptide, or otheroligomer; or natural or non-natural steroid:

-   X₁, X₂, and X₃ are independently oxygen, methylene,    monochloromethylene, dichloromethylene, monofluoromethylene,    difluoromethylene, or imido;-   T₁, T₂, W, and V are independently oxygen or sulfur;-   m=0,1 or 2;-   n=0 or 1;-   p=0,1, or 2;-   where the sum of m+n+p is from 0 to 5;-   M=H or a pharmaceutically-acceptable inorganic or organic counter    ion;-   D=O or CH₂;-   B is a purine or a pyrimidine residue according to general Formulae    IV and V which is linked to the 1′ position of the furanose or    carbocycle via the 9- or 1- position of the base, respectively;-   Y═H, OH, or OR₄;-   Z═H, OH, or OR₅; with the proviso that Y and Z are not both H;-   R₄ and R₅ are residues which are linked directly to the 2′ and /or    3′ oxygens of the furanose or carbocycle via a carbon atom according    to Formula II, or linked directly to the two 2′ and 3′ oxygens of    the furanose or carbocycle via a common carbon atom according to    Formula III.

The present invention is also directed to a method of preventing,diagnosing or treating epithelial diseases or conditions; such diseasesinclude respiratory diseases, eye diseases, vaginal and cervicaldryness, gastrointestinal tract diseases, inflammatory and allergicdiseases, such as chronic bronchitis, cystic fibrosis, sinusitis, lungcancer, otitis media, retinal detachment, retinal edema, dry mouth,gastroesophageal reflux disease (GERD), constipation, glaucomaassociated with elevated intraocular pressure, retinal degenerativediseases, corneal edema, allergic conjunctivitis, ocular surfaceinflammation, allergic rhinitis. A further aspect of the presentinvention is directed to a method of preventing or treating diseases ofthe joint; such diseases include osteoarthritis and rheumatoidarthritis. Yet a further aspect of the present invention is directed toa method of preventing or treating diseases associated with plateletaggregation and thrombosis in humans and other mammals.

The method comprises administering to a subject a pharmaceuticalcomposition comprising a therapeutically effective amount of anucleotide of Formula I, wherein said amount is effective to diagnose,prevent, or treat such epithelial tissue diseases.

The present invention is also directed to a method of preventing ortreating epithelial diseases or conditions associated therewith, themethod comprises:

-   -   (a) identifying a mammal at risk for epithelial tissue diseases;        and    -   (b) applying a composition comprising a compound of Formula I in        an amount effective to prevent or treat epithelial tissue        diseases or conditions associated here within.

The present invention is also directed to a method of preventingepithelial tissue diseases or conditions associated therewith, themethod comprises:

-   -   (a) applying to a mammal at risk for epithelial tissue diseases        a composition comprising a compound of Formula I in an amount        effective to prevent the incidence of epithelial tissue        diseases.    -   (b) determining whether such disease or condition developed.

The invention also provides novel pharmaceutical compositions comprisingcompounds of Formula I in a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

The applicants have unexpectedly discovered that the benefits of adinucleotide pharmaceutical can be achieved by a mononucleotide that hasbeen modified by attaching a degradation resistant substituent A on theterminal phosphate of a nucleoside polyphosphate. The pharmacologicalactivity of the mononucleotide is unexpectedly maintained, and in someinstances enhanced, when this degradation resistant substituent ispresent. Further, by attaching this degradation resistant substituent,the stability from degradation matches or exceeds those of certaindinucleotides. The applicants have unexpectedly discovered that certainbenefits of a dinucleotide can be obtained even when one end of adinucleotide is replaced by degradation resistant substituent that doesnot have the activity of a nucleotide. In the worst case, this newmononucleotide molecule will only have half of the efficacy of thecomparable dinucleotide, but in many instances that lower efficacy iscompletely acceptable, particularly when viewing the benefits of the newmolecule.

In many instances the degradation resistant substituent can have its ownpharmacological activity, different from those of nucleotides. Further,these new molecules, due to the degradation resistant substituent A, inmany instances have the benefits of 1) ease in manufacture, e.g.superior physical chemical characteristics which lend to simplifiedpurification schemes; 2) reduced costs, as nearly all of thesubstituents described as A are less costly than nucleosides; 3) fewerstereochemistry concerns as few substituents are as stereochemicallycomplex as nucleosides; 4) enhanced pharmacokinetic properties asnon-nucleoside substituents can possess a myriad of differingcharacteristics; and/or 5) enhanced chemical stability as nucleosidesare inherently less stable than most organic molecules.

Important criteria for these new molecules are stability and that thedegradation resistant substituent does not interfere with the activityof the nucleotide. This means that the degradation resistant substituentis no larger than 1000 Daltons, preferably less than 500, and that thesubstituent does not adversely affect pharmacological activity ortoxicity or alternatively is beneficial. Further, this degradationresistant substituent on the nucleotide must not react with othernucleotide molecules or with other components of the pharmaceuticalformulation in ways that would detrimentally modify the nucleotide'spharmacological activity. This means that this degradation resistantsubstituent must be reasonably stable within a pharmaceuticalformulation.

The present invention provides compounds of Formula I as well as methodsof preventing, diagnosing or treating epithelial and retinal tissuediseases or conditions. The method comprises administering to a subjecta pharmaceutical composition comprising a therapeutically effectiveamount of the compound of general Formula I and pharmaceuticallyacceptable salts thereof:

wherein:A is a covalently bound degradation resistant substituent that has amolecular weight of no more than about 1000 and is OR₁, SR₁, NR₁R₂, orCR₁R₂R₃ such that R₁, R₂, and R₃ are independently hydrogen, alkyl,cycloalkyl, aryl, arylalkyl, phosphonate, or acylthioalkyl, with orwithout substituents or heteroatoms, or taken together to form acycloalkyl or aryl ring, with or without substituents or heteroatoms,with the exception of OR₁ and SR₁ not being OH or SH; or a natural ornon-natural amino acid, peptide, polypeptide, or other oligomer; ornatural or non-natural steroid. In other words, A is a covalently boundsubstituent having a maximum molecular weight of 1000 and selected fromthe group consisting of a natural or non-natural amino acid, a peptide,a polypeptide, an oligonucleotide, a polynucleotide, a natural ornon-natural steroid. A is preferably a hydroxylated alkyl group (e.g.glycerol, cholesterol); is an amino acid (e.g. phenylalanine, serine,tyrosine) having 3 to 50 carbon atoms; is amino or mono- ordisubstituted amino, where the substituents are alkyl, cycloalkyl,aralkyl, aryl, substituted aralkyl, or substituted aryl having 3 to 50carbon atoms and which may also contain heteroatoms (e.g. S, N, O).

-   X₁, X₂, and X₃ are independently oxygen, methylene,    monochloromethylene, dichloromethylene, monofluoromethylene,    difluoromethylene, or imido;-   T₁, T₂, W, and V are independently oxygen or sulfur;-   m=0 , 1 or 2;-   n=0 or 1;-   p=0, 1,or 2;-   where the sum of m+n+p is from 0 to 5 (preferably 2 or 3);-   M=H or a pharmaceutically-acceptable inorganic or organic counter    ion;-   D=O or CH₂;-   B is a purine or a pyrimidine residue according to general Formulae    IV and V which is linked to the 1′ position of the furanose or    carbocycle via the 9- or 1 - position of the base, respectively;-   Y═H, OH, or OR₄;-   Z═H, OH, or OR₅; with the proviso that Y and Z are not both H;-   R₄ and R₅ are residues which are linked directly to the 2′ and /or    3′ oxygens of the furanose or carbocycle via a carbon atom according    to Formula II, or linked directly to the two 2′ and 3′ oxygens of    the furanose or carbocycle via a common carbon atom according to    Formula III.

wherein:

-   O is the corresponding 2′ and/or 3′ oxygen of the furanose or    carbocycle;-   R₆, R₇, and R₈ are H, an alkyl, cycloalkyl, aralkyl, aryl,    substituted aralkyl, or substituted aryl, such that the moiety    defined according to Formula II is an ether; or-   R₆ and R₇ are H, an alkyl, cycloalkyl, aralkyl, aryl, substituted    aralkyl, or substituted aryl, and R₈ is alkoxy, cycloalkoxy,    aralkyloxy, aryloxy, substituted aralkyloxy, or substituted aryloxy    such that the moiety defined according to formula II is an acyclic    acetal or ketal; or-   R₆ and R₇ are taken together as oxygen or sulfur doubly bonded to C,    and R₈ is alkyl, cycloalkyl, aralkyl, aryl, substituted aralkyl, or    substituted aryl, such that the moiety defined according to Formula    II is an ester or thioester; or-   R₆ and R₇ are taken together as oxygen or sulfur doubly bonded to C,    and R₈ is amino or mono- or disubstituted amino, where the    substituents are alkyl, cycloalkyl, aralkyl, aryl, substituted    aralkyl, or substituted aryl, such that the moiety according to    Formula II is a carbamate or thiocarbamate; or-   R₆ and R₇ are taken together as oxygen or sulfur doubly bonded to C,    and R₈ is alkoxy, cycloalkoxy, aralkyloxy, aryloxy, substituted    aralkyloxy, or substituted aryloxy, such that the moiety according    to Formula II is a carbonate or thiocarbonate; or-   R₈ is not present and R₆ and R₇ are taken together as oxygen or    sulfur doubly bonded to C and both the 2′ and 3′ oxygens of the    furanose are directly bound to C to form a cyclical carbonate or    thiocarbonate;

wherein:

-   O is the 2′ and 3′ oxygens of the furanose or carbocycle; and the 2′    and 3′ oxygens of the furanose or carbocycle are linked by a common    carbon atom (C) to form a cyclical acetal, cyclical ketal, or    cyclical orthoester;-   for cyclical acetals and ketals, R₉ and R₁₀ are independently    hydrogen, alkyl, cycloalkyl, aralkyl, aryl, substituted aralkyl,    substituted aryl or can be joined together to form a homocyclic or    heterocyclic ring composed of 3 to 8 atoms, preferably 3 to 6 atoms;    for cyclical orthoesters, R₉ is hydrogen, alkyl, cycloalkyl,    aralkyl, aryl, substituted aralkyl, or substituted aryl, R₁₀ is    alkyloxy, cycloalkyloxy, aralkyloxy, aryloxy, substituted    aralkyloxy, or substituted aryloxy;

wherein:

-   R₁₁ and R₁₅ are hydroxy, oxo, amino, mercapto, alkylthio, arylthio,    alkyloxy, aryloxy, alkylamino, cycloalkylamino, aralkylamino,    arylamino, diaralkylamino, diarylamino, or dialkylamino, where the    alkyl groups are optionally linked to form a heterocycle; or-   R₁₁ and R₁₅ are acylamino, provided that they incorporate an amino    residue from the C-6 position of the purine or the C-4 position of    the pyrimidine; or-   when R₁₁ in a purine or R₁₅ in a pyrimidine has as its first atom    nitrogen, R₁₁ and R₁₂ or R₁₅ and R₁₆ are taken together to form a    5-membered fused imidazole ring (etheno compounds), optionally    substituted on the etheno ring with alkyl, cycloalkyl, aralkyl, or    aryl moieties, as described for R₆–R₁₀ above; or-   when R₁₅ in a pyrimidine has as its first atom oxygen, R₁₅ and R₁₇    are taken together to form a 5-membered dihydrofuran ring,    optionally substituted on the dihydrofuran ring with alkyl,    cycloalkyl, aralkyl, or aryl moieties, as described for R₆–R₁₀    above;-   J is carbon or nitrogen, with the provision that when nitrogen, R₁₃    is not present;-   R₁₂ is hydrogen, O (adenine 1-oxide derivatives) or is absent    (adenine derivatives);-   R₁₆ is hydrogen, or acyl (e.g. acetyl, benzoyl, phenylacyl, with or    without substituents);-   R₁₃ is hydrogen, alkyl, bromo, azido, alkylamino, arylamino or    aralkylamino, alkoxy, aryloxy or aralkyloxy, alkylthio, arythio or    aralkylthio, or ω-E(C₁₋₆alkyl)G-, wherein E and G are independently    amino, mercapto, hydroxy or carboxyl;-   R₁₄ is hydrogen, halo, amino, monosubstituted amino, disubstituted    amino, alkylthio, arylthio, or aralkylthio, where the substituent on    sulfur contains up to a maximum of 20 carbon atoms, with or without    unsaturation;-   R₁₇ is hydrogen, methyl, alkyl, halo, alkyl, alkenyl, substituted    alkenyl, alkynyl, or substituted alkynyl.

Compounds according to Formulae IV and V where R₁₁ or R₁₅ is acylaminofor the most part fall within the scope of Formula VI:

wherein:

-   NH is the amino residue at the C-6 position in a purine or the amino    residue at the C-4 position in a pyrimidine;-   W is oxygen or sulfur;-   R₁₈ is amino or mono- or disubstituted amino such that the moiety    according to Formula VI is a urea or thiourea; or R₁₈ is alkoxy,    aralkyloxy, aryloxy, substituted aralkyloxy, or substituted aryloxy,    such that the moiety according to Formula VI is a carbamate or    thiocarbamate; or-   R₁₈ is alkyl, cycloalkyl, aralkyl, or aryl, with or without    substituents or heteroatoms, such that the moiety according to    Formula VI is an amide; with definitions of alkyl, cycloalkyl,    aralkyl, or aryl groups as previously defined for comparable groups    in R₆ to R₁₀.-   The following applies to all of the R groups above: The number of    carbon atoms for the alkyl substituent preferably ranges from 1 to    50 carbons, however, the more preferred alkyl range is 2 to 25    carbons, even more preferred is 3 to 15 carbons, and most preferred    is 4 to 10 carbons. The number of carbon atoms for the cycloalkyl    substituent preferably ranges from 5 to 30 carbons, however, the    more preferred cycloalkyl range is 7 to 30 carbons, even more    preferred is 7 to 20 carbons, and most preferred is 8 to 15 carbons.    The preferred aralkyl range is 7 to 30 carbons, more preferred is 7    to 20 carbons, and most preferred is 8 to 15 carbons. The preferred    aryl range is 7 to 30 carbons, more preferred is 7 to 20 carbons,    and most preferred is 8 to 15 carbons. The preferred alkoxy range is    1 to 25 carbons, more preferred is 1 to 18 carbons, and most    preferred is 1 to 10 carbons. The preferred cycloalkoxy range is 6    to 30 carbons, more preferred is 6 to 20 carbons, and most preferred    is 6 to 15 carbons. The preferred aralkyloxy range is 7 to 30    carbons, more preferred is 7 to 20 carbons, and most preferred is 8    to 15 carbons. The preferred aryloxy range is 6 to 30 carbons, more    preferred is 6 to 20 carbons, and most preferred is 8 to 15 carbons.    The preferred alkylthio range is 1 to 25 carbons, more preferred is    1 to 18 carbons, and most preferred is 1 to 10 carbons. The    preferred aralkylthio range is 7 to 30 carbons, more preferred is 7    to 20 carbons, and most preferred is 8 to 15 carbons. The preferred    arylthio range is 6 to 30 carbons, more preferred is 6 to 20    carbons, and most preferred is 8 to 15 carbons. The preferred    alkenyl range is 2 to 25 carbons, more preferred is 2 to 20 carbons,    and most preferred is 3 to 15 carbons. The preferred alkynyl range    is 3 to 25 carbons, more preferred is 3 to 15 carbons, and most    preferred is 4 to 12 carbons. The heteroatoms are sulfur, oxygen,    nitrogen, phosphorous, boron, and silicon; the halogens are    fluorine, chlorine, bromine, and iodine; and the amino acids    include, but are not limited to, both D- and L- forms of alanine,    arginine, aspartic acid, cysteine, cystine, glutamic acid,    glutamine, glycine, histidine, isoleucine, leucine, lysine,    methionine, ornithine, phenylalanine, proline, serine, threonine,    tryptophan, tyrosine, and valine.

One general synthetic scheme for the synthesis of compounds of theinvention employs activation of a nucleoside mon-, di-, or triphosphatewith an activating agent such as carbonyldiimidazole, phosphorous oxychloride, etc. and subsequent reaction with a nucleophile, Nu (e.g.R₁OH, R₁SH, NHR₁R₂, etc.), to the activated terminal phosphate moiety. Bis any purine or pyrimidine, natural or synthetic The product is shownas a ribofuranosyl sugar in the β-D configuration for illustrationpurposes only, and is not intended to be limiting in scope.

Alternatively, compounds of the invention can be made according to thescheme below in which a nucleoside mono-, di-, or triphosphate is addedto an electrophile, El (e.g. activated sugar, activated carboxylic acid,activated carbon, activated amino acid, etc.). B is any purine orpyrimidine, natural or synthetic. The product is shown as aribofuranosyl sugar in the β-D configuration for illustration purposesonly, and is not intended to be limiting in scope.

The compounds of the present invention can be conveniently synthesizedby those skilled in the art using well-known chemical procedures.5′-nucloside mono-, di-, tri-, and tetraphosphates can be obtained fromcommercial sources or can be synthesized from the nucleoside using avariety of phosphorylation reactions, which can be found in the chemicalliterature. Nucleoside mono- and diphosphates so obtained can be reactedwith carbonyldiimidazole, dicyclohexylcarbodiimide or other suitableactivating reagents, and coupled with a variety of nucleophiles toinstall unique substituents on the terminal phosphate. Activation ofnucleoside triphosphates with dicyclohexylcarbodiimide gives a cyclicaltrimetaphosphate as the activated species, which can be advantageouslyring opened with nucleophiles, to give substituents on the terminal (γ)phosphate of the triphosphate. If the cyclical trimetaphosphate isopened with reagents containing phosphate as the nucleophile, nucleoside5′-tetraphosphates are produced with novel moieties on the terminal (δ)phosphate. Alternately, these same phosphate nucleophiles can be reactedwith the previously described activated nucleoside mono- anddiphosphates, giving di- and triphosphates (respectively) that fallwithin the scope of the present invention.

As mentioned above, the role of the phosphate chain can be reversed suchthat the terminal phosphate of the chain can serve as a nucleophiletowards electrophilic reagents. Examples of electrophilic reagentsinclude alkyl and aralkyl halides and sulfonates, activated acylcompounds, activated phosphorous compounds, and the like.

For the compounds of the present invention which are modified on thenucleic acid base or furanose in addition to the phosphate chain, themodifications can be made at the level of the nucleoside, followed byphosphorylation and condensation with nucleophiles as previouslydescribed, or the reactions can be carried out directly on thepreassembled nucleotide. In general Formula I, the substituents at Y andZ can be ethers, esters, acyclic acetals and ketals, carbamates, orcarbonates, which are generally described by Formula II. Ethers can beprepared by reacting a hydroxyl group in a nucleoside or nucleotide withan activated form of an appropriate alkyl or aralkyl, such as analkyl/aralkyl halide, alkyl/aralkyl sulfonate and the like, usually inthe presence of an organic or inorganic base. Esters can be readilyprepared by reacting a hydroxyl group in a nucleoside or nucleotide withan activated form of an appropriate organic acid, such as an acid halideor acid anyhydride in the presence of an organic or inorganic base.Alternately, use of a suitable coupling reagent such asdicyclohexylcarbodiimide, 1,1′-carbonyldiimidazole and the like toactivate the organic acid can be used to achieve the same result.Acyclic acetals and ketals can be prepared by the reaction between asingle hydroxyl in a nucleoside or nucleotide with aldehydes or ketones(respectively) or their chemical equivalents, under acidic conditions.

Carbamates or thiocarbamates can be most conveniently prepared byreaction of a hydroxyl group in a nucleoside or nucleotide with any of anumber of commercially available isocyanates or isothiocyanates,respectively, in an inert solvent. Carbonates or thiocarbonates can besynthesized by reacting the hydroxyl groups in a nucleoside ornucleotide with an appropriate haloformate in the presence of an organicor inorganic base.

In the general Formula I, the substituents at Y and Z, when takentogether, can be taken to mean acetals, ketals or orthoesters, asdescribed by Formula III. Acetals and ketals can be readily prepared byreaction of the neighboring 2′ and 3′ hydroxyl groups in an appropriatenucleoside or nucleotide with an aldehyde or ketone, respectively, ortheir chemical equivalents, in the presence of an acid catalyst. Typicalacids include trichloroacetic, p-toluenesulfonic, and methanesulfonicemployed in catalytic amounts, in conjunction with inert solvents.Alternately, weaker organic acids such as formic can be used as both thecatalyst and solvent for the reaction.

Cyclical orthoesters can be prepared by reaction of the neighboring 2′and 3′ hydroxyl groups in a nucleoside or nucleotide with an acylicorthoester, in the presence of an acid.

When the nucleoside or nucleotide to be derivatized is a purine thatcontains a 6-amino functionality or is a pyrimidine that contains a4-amino functionality, it can be converted to the respective urea orthiourea, as described by general formula VI. This can be accomplishedby treatment with isocyanates or isothiocyanates, respectively, as waspreviously described for carbamates or thiocarbamates of the 2′ or 3′hydroxyls. Reactions of these amino groups with isocyanates orisothiocyanates can be carried out in the presence of the unprotectedhydroxyl groups, by appropriate manipulation of the stoichiometry of thereaction.

Those skilled in the art will recognize various synthetic methodologieswhich can be employed to prepare non-toxic pharmaceutically acceptablesalts and acylated prodrugs of the compounds of the present invention.Methods of preparing these from the compound of Formula I includepassing an aqueous solution through a column of ion exchange resin inthe desired cation form, thus converting the compound to the desiredsalt form. If the desired end product is a sodium salt, such as A onuridine tetraphosphate tetrasodium salt, the starting material (anammonium or other salt) is passed through a DOW 50 H+ column toprotonate the compound and generate the free acid. This protonatedcompound is collected in an aqueous solution of sodium hydroxide whichforms the sodium salt.

As is typical for nucleotide chemistry, the reactions which give rise tocompounds of the present invention usually end with several productsbeing formed, owing to multiple reactive sites in these molecules. Whenmultiple products are obtained, these can be separated by the use ofpreparative reverse phase high performance liquid chromatography (HPLC).Particularly advantageous is the use of C18 or phenyl reverse phasecolumns, in conjunction with gradients that start with ammonium acetatebuffer and end with methanol. Following chromatography, the products areisolated by evaporation of the solvent, followed by lyophilization.

While separation of multiple products can be done by HPLC, anotherstrategy is to use nucleosides or nucleotides which contain only asingle functionality which is reactive under the conditions beingemployed. This can be accomplished by the use of protecting groups toblock side reactions at other positions in the molecule. This can bedone at the level of the nucleoside prior to phosphorylation andcoupling of the phosphate chain with a nucleophile, or at the level ofthe nucleotide.

The second aspect of the present invention provides methods ofpreventing, diagnosing or treating epithelial and retinal tissuediseases or conditions. The method comprises administering to a subjecta pharmaceutical composition comprising a therapeutically effectiveamount of the compound of general Formula I and pharmaceuticallyacceptable salts thereof.

The present invention is also directed to a method of preventing ortreating diseases or conditions associated with enhancing clearance ofsecretions in the respiratory tract by increasing the hydration ofretained mucus secretions, stimulating the production of mucins, andincreasing ciliary beat frequency. Prevention or treatment of diseasesthat could benefit from enhancing clearance of secretions by increasingthe hydration of retained mucus secretions, stimulating the productionof mucins, and increasing ciliary beat frequency are chronic obstructivepulmonary diseases such as chronic bronchitis, acute bronchitis, acuteexacerbations of chronic bronchitis, PCD, cystic fibrosis, as well asprevention of pneumonia due to immobility. Furthermore, because of theirgeneral ability to clear retained mucus secretions and stimulate ciliarybeat frequency, the compounds of the present invention are also usefulin the treatment of acute and chronic sinusitis and otitis media inmammals, including humans. By enhancing secretion clearance, thecompounds are useful as protection before or after exposure to inhaledbiological warfare agents. They can also be used to enhance lung imagingby clearing secretions from the lungs prior to obtaining the image, fordetection of lung disease through an increase in sputum production. Themethod comprises administering to a subject a pharmaceutical compositioncomprising a therapeutic effective amount of a nucleotide, wherein saidamount is effective to hydrate the mucosal membranes of the respiratorytract.

Still further indications where the compounds of the invention areuseful are for a method of stimulating cervical and vaginal secretionsin a subject in need of such treatment. The method of the presentinvention can be used to increase cervical and vaginal secretions forany reason, including, but not limited to, treatment of vaginal drynessand/or treatment of vulvar pain. Vaginal dryness is associated with butnot limited to menopause, childbirth, breastfeeding, chemotherapy orradiotherapy, diabetes mellitus, Sjögren's syndrome, Ehlers-Danlossyndrome, systemic sclerosis and other systemic autoimmune diseases,hysterectomy, urogenital surgery, psychosomatic disorders, anxiety,psychosexual problems, and pharmacological drug-related side effects.The method comprises administering to a subject a pharmaceuticalcomposition comprising a therapeutic effective amount of a nucleotide,wherein said amount is effective to hydrate the mucosal membranes in thevaginal and cervical tracts.

Still further indications where the compounds of the invention areuseful are for a method of regulating mucus secretions and fluidtransport in the gastrointestinal system of a mammal, including humans.There are many situations where it is therapeutically desirable toincrease the amount of mucin secretion, bicarbonate secretions, and/ordegree of hydration in gastrointestinal systems. When the mucosalbarrier is impaired in the digestive tract, it results in diseases suchas dry mouth, gastro-esophageal reflux disease, peptic ulcer,inflammatory bowel disease, etc. Abnormal fluid and electrolytictransport in the lower gastrointestinal tract results in disorders suchas constipation and diarrhea. Proper regulation of fluid andelectrolytic absorption and secretion at appropriate regions along thegastrointestinal system is required for normal digestive function. Theinvention provides a method of regulating mucus/mucin secretions, andfluid transport in the gastrointestinal tract. The invention provides amethod for treating gastrointestinal disease in which the mucosalbarrier of the gastrointestinal system is impaired. Gastrointestinaldiseases suitable for treatment by this invention include diseases ordisorders affecting the buccal cavity (primary salivary), esophagus,stomach, small intestine, large intestine, rectum and ancillary organssuch as pancreas, liver and gall bladder. The invention additionallyprovides a method for correcting disorders of fluid secretion orabsorption in the gastrointestinal. For example, dry mouth, mouth ulcer,gum disease, esophageal reflux disease, peptic ulcer, inflammatory boweldisease (ulcerative colitis and Crohn's disease), diarrhea andconstipation can be treated by the present method. In addition,gastrointestinal problems associated with cystic fibrosis diseases suchas dry mucin and decreased absorption of nutrient by epithelial cells inthe gastrointestinal tract can also be treated by the present method. Inaddition, gastrointestinal problems caused by cancer and chemotherapycan also be treated by this method. The method comprises administeringto a subject a pharmaceutical composition comprising a therapeuticeffective amount of a compound of Formula I or a pharmaceuticallyacceptable salt thereof, wherein said amount is effective to hydrate themucosal membranes of the gastrointestinal tract.

The present invention is also directed to a method of preventing ortreating diseases or conditions associated with the ocular surface. Suchconditions of the ocular surface include, but not limited to, dry eyedisease and ocular surface inflammation. This method for treatment ofthe causes of dry eye disease is through stimulating tear secretionsfrom conjunctival tissues. The present invention is also directed to amethod of preventing or treating ocular surface inflammation as well asother eye related conditions such as keratoconjunctivitis sicca (KCS),age-related dry eye, Stevens-Johnson syndrome, Sjögren syndrome; ocularcicatrical, pemphigoid; bletharitis; corneal injury; infection;Reilly-Day syndrome; congenital alacrima; nutritional disorders;pharmacologic side-effects; eye stress and glandular and tissue; smogexposure; smoke exposure; dry air caused by insufficient hydration ofthe ocular surface.

Still further indications where the compounds of the invention areuseful are for the treatment of other diseases or conditions associatedwith the mammalian eye. Degenerative retinopathies generally affect twoneuronal cell populations in the retina: the photoreceptors and ganglioncells. Glial cells in the mature nervous systems provide trophic supportto neurons and are therefore a viable cellular target to effect neuronalpreservation and survival in a variety of neurodegenerative conditions.This invention is also directed to a method for treating diseases orconditions associated with retinal degeneration, removal of fluid inretinal detachment and retinal edema as well as treatment of ocularhypertension. Retinal degeneration is often an endpoint of a variety ofocular and systemic diseases and environmental conditions, such asmacular degeneration, glaucoma, retinitis pigmentosa, optic nervedegeneration, optic neuritis, chronic metabolic diseases (diabeticretinopathy) neurotoxins, ischemia and physical trauma.

The methods and compositions disclosed in the present invention can beused to stimulate removal of extraneous intra-retinal or subretinalfluid for any reason, including, but not limited to, primary andadjunctive treatments of rhegmatogenous retinal detachment, serousretinal detachment, all forms of cystoid macular edema (uveitis,post-surgical, central and branch vein occlusion, and inherited retinaldiseases such as retinitis pigmentosa), and all forms of retinal andmacular edema (proliferative and non-proliferative, exudativeage-related macular degeneration, and retinopathy of prematurity). Themethod comprises administering to a subject a pharmaceutical compositioncomprising a therapeutic effective amount of a compound of Formula I ora pharmaceutically acceptable salt thereof, wherein said amount iseffective in said treatment.

Still further indications where the compounds of the invention areuseful in the management and/or treatment of primary glaucoma, whichconsists of two types: narrow angle or acute congestive and wide angleor chronic simple glaucoma. Yet another embodiment of the presentinvention is the management of secondary glaucoma.

The present invention provides a method of treating and/or managingglaucoma, by facilitating the outflow of fluid from the eye and therebyreduce the accumulation of said fluid contributing to increasedintraocular pressure characteristic of glaucoma. The method comprisesco-administration to a subject, an effective dose of a pharmaceuticalcomposition comprising a purinergic receptor ligand, with or withouttherapeutic and adjuvant agents commonly used to treat or manageglaucoma.

The present invention is also directed to a method of stimulating thesecretion of synovial fluid, mucins, hyaluronic acid, and/or surfaceactive phospholipids, and thereby enhancing joint lubrication, using anucleotide in patients in need of such treatment. This is also a methodof enhancing joint lubrication comprising: administering to a subject inneed of such treatment a purinergic receptor agonist in an amounttherapeutically effective to enhance joint lubrication. A method oftreating osteoarthritis comprising a therapeutic effective amount of aP2Y receptor agonist ligand, in an appropriate pharmaceuticalcomposition, administering to a subject in need of enhancing jointlubrication in an amount therapeutically effective to treatosteoarthritis.

The present invention is also directed to a method preventing and/orreversing the symptoms and manifestations of inflammatory diseases, andhence a method of treating inflammation.

Still further indications where the compounds of the invention areuseful are for a method of preventing and/or reversing the symptoms andmanifestations of allergic reactions and thus a method of treatingallergies.

This method comprises administering to a subject in need thereof apharmacological composition comprising a compound of Formula I or apharmaceutically acceptable salt thereof in an amount effective totreat, prevent, and/or reverse the symptoms and manifestations ofinflammatory diseases, together with a pharmaceutically acceptablecarrier.

The present invention is also directed to a method of preventing ortreating diseases or conditions associated with platelet aggregation.The method is also directed to a method of treating thrombosis in humansand other mammals. An intravascular thrombus results from a pathologicaldisturbance of hemostasis. Platelet adhesion and aggregation arecritical events in intravascular thrombosis. There is a need in the areaof cardiovascular and cerebrovascular therapeutics for an agent that canbe used in the prevention and treatment of thrombi, with minimal sideeffects, such as unwanted prolongation of bleeding in other parts of thecirculation, while preventing or treating target thrombi. This inventionis also directed to a method of preventing or treating diseasesassociated with platelet aggregation. Such related diseases are, but notrestricted to, thrombosis, primary arterial thrombotic complications ofatherosclerotic disease; thrombotic complications of surgical ormechanical damage; mechanically induced platelet aggregation; shuntocclusion; thrombosis secondary to vascular damage and inflammation;indications with a diffuse thrombotic/platelet composition component;venous thrombosis; coronary arterial thrombosis; pathological effects ofatherosclerosis and arterial sclerosis, chronic or acute states of hyperaggregability, reocclusion of an artery or vein following fibrinolytictherapy, platelet adhesion associated with extracorporeal circulation,thrombotic complications associated with thrombolytic therapy,thrombotic complications associated with coronary and other angioplasty,and thrombotic complications associated with coronary artery bypassprocedures, venous thrombosis, thrombophiebitis, arterial embolism,coronary and cerebral arterial thrombosis, unstable angina, coronaryangioplasty, myocardial infarction, cerebral embolism, kidney embolismsand pulmonary embolisms. Primary arterial thrombotic complications ofatherosclerotic disease include angioplasty, endarterectomy, stentplacement, coronary and other vascular graft surgery. Thromboticcomplications of surgical or mechanical damage include tissue salvagefollowing surgical or accidental trauma, reconstructive surgeryincluding skin flaps, and “reductive” surgery such as breast reduction.Mechanically-induced platelet activation is caused by cardiopulmonarybypass resulting in microthromboembolism or caused by storage of bloodproducts. Shunt occlusion occurs in renal dialysis and plasmapheresis.Thrombosis secondary to vascular damage and inflammation includevasculitis, arteritis, glomerulonephritis and organ graft rejection. Theindications with a diffuse thrombotic/platelet consumption componentinclude disseminated intravascular coagulation, thromboticthrombocytopenic purpura, hemolytic uremic syndrome, heparin-inducedthrombocytopenia, and pre-eclampsia/eclampsia. The Venous thrombosisincludes deep vein thrombosis, veno-ocelusive disease, hematologicalconditions, and migraine. Hematological conditions includethrombocythemia and polycythemia. Coronary arterial thrombosis isassociated with unstable angina, coronary angioplasty and acutemyocardial infarction. Pathological effects of atherosclerosis andarteriosclerosis include arteriosclerosis, acute myocardial infarction,chronic stable angina, unstable angina, transient isehemic attacks,strokes, peripheral vascular disease, arterial thrombosis, preeclampsia,embolism, restenosis or abrupt closure following angioplasty, carotidendarterectomy, and anastomosis of vascular grafts. Chronic or acutestates of hyper-aggregability are caused by DIC, septicemia, surgical orinfectious shock, post-operative and post-partum trauma, cardiopulmonarybypass surgery, incompatible blood transfusion, abruptio placentae,thrombotic thrombocytopenic purpura, snake venom and immune diseases.Reocclusion of an artery or vein following fibrinolytic therapy isinhibited by internal administration of said compound with afibrinolytic agent. The fibrinolytic agent is selected from the groupconsisting of natural or synthetic products, which directly orindirectly cause lysis of a fibrin clot. The fibrinolytic agent is aplasminogen activator selected from the group consisting ofanistreplase, urokinase (UK), pro-urokinase (PUK), streptokinase (SK),tissue plasminogen activator (tPA) and mutants, or variants thereof,which retain plasminogen activator activity. The variants are selectedfrom a group consisting of variants which have been chemically modified,variants which one or more amino acids have been added, deleted orsubstituted or variants with one or more modified functional domains.The modified functional domains are added, deleted or altered bycombining the active site of one plasminogen activator or fibrin bindingdomain of another plasminogen activator or fibrin binding molecule.Still further indications where the compounds of the invention areuseful are for the prevention of platelet aggregation and clot formationin blood and blood products during storage.

The compounds of the present invention also encompass their non-toxicpharmaceutically acceptable salts, such as, but not limited to, analkali metal salt such as lithium, sodium or potassium; an alkalineearth metal salt such magnesium or calcium; or an ammonium or tetraalkylammonium salt, i.e., NX₄ ⁺(wherein X is C₁₋₄). Pharmaceuticallyacceptable salts are salts that retain the desired biological activityof the parent compound and do not impart undesired toxicologicaleffects.

Compounds of the present invention can be administered systemically totarget sites in a subject in need such that a target dose in the rangeof 10⁻¹ to 10⁻⁶ M is achieved and preferably in the range of 10⁻² to10⁻⁴ M.

For systemic administration such as injection and infusion, thepharmaceutical formulation is prepared in a sterile medium. The activeingredient, depending on the vehicle and concentration used, can eitherbe suspended or dissolved in the vehicle. Adjuvants such as localanesthetics, preservatives and buffering agents can also be dissolved inthe vehicle. The sterile indictable preparation can be a sterileindictable solution or suspension in a non-toxic acceptable diligent orsolvent. Among the acceptable vehicles and solvents that can be employedare sterile water, saline solution, or Ringer's solution.

Another method of systemic administration of the active compoundinvolves oral administration, in which pharmaceutical compositionscontaining active compounds are in the form of tablets, lozenges,aqueous or oily suspensions, viscous gels, chewable gums, dispersiblepowders or granules, emulsion, hard or soft capsules, or syrups orelixirs.

For oral use, an aqueous suspension is prepared by addition of water todispersible powders and granules with a dispersing or wetting agent,suspending agent one or more preservatives, and other excipients.Suspending agents include, for example, sodium carboxymethylcellulose,methylcellulose and sodium alginate. Dispersing or wetting agentsinclude naturally-occurring phosphatides, condensation products of anallylene oxide with fatty acids, condensation products of ethylene oxidewith long chain aliphatic alcohols, condensation products of ethyleneoxide with partial esters from fatty acids and a hexitol, andcondensation products of ethylene oxide with partial esters derived fromfatty acids and hexitol anydrides. Preservatives include, for example,ethyl, and n-propyl p-hydroxybenzoate. Other excipients includesweetening agents (e.g., sucrose, saccharin), flavoring agents andcoloring agents. Those skilled in the art will recognize the manyspecific excipients and wetting agents encompassed by the generaldescription above.

For oral application, tablets are prepared by mixing the active compoundwith nontoxic pharmaceutically acceptable excipients suitable for themanufacture of tablets. These excipients can be, for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for example,starch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate can be employed. Formulations fororal use can also be presented as hard gelatin capsules wherein theactive ingredient is mixed with an inert solid diluent, for example,calcium carbonate, calcium phosphate or kaolin, or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, for example, peanut oil, liquid paraffin or olive oil.Formulation for oral use can also be presented as chewable gums byembedding the active ingredient in gums so that the active ingredient isslowly released upon chewing.

Additional means of systemic administration of the active compound tothe target platelets of the subject would involve a suppository form ofthe active compound, such that a therapeutically effective amount of thecompound reaches the target sites via systemic absorption andcirculation.

For rectal administration, the compositions in the form of suppositoriescan be prepared by mixing the active ingredient with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the compound. Such excipients include cocoa butter andpolyethylene glycols.

The active compounds can also be systemically administered to the sitesthrough absorption by the skin using transdermal patches or pads. Theactive compounds are absorbed into the bloodstream through the skin.Plasma concentration of the active compounds can be controlled by usingpatches containing different concentrations of active compounds.

One systemic method involves an aerosol suspension of respirableparticles comprising the active compound, which the subject inhales. Theactive compound would be absorbed into the bloodstream via the lungs,and subsequently contact the target in a pharmaceutically effectiveamount. The respirable particles can be liquid or solid, with a particlesize sufficiently small to pass through the mouth and larynx uponinhalation; in general, particles ranging from about 1 to 10 microns,but more preferably 1–5 microns, in size are considered respirable.

Another method of systemically administering the active compounds to theplatelet aggregation sites of the subject involves administering aliquid/liquid suspension in the form of eye drops or eye wash or nasaldrops of a liquid formulation, or a nasal spray of respirable particlesthat the subject inhales. Liquid pharmaceutical compositions of theactive compound for producing a nasal spray or nasal or eye drops can beprepared by combining the active compound with a suitable vehicle, suchas sterile pyrogen free water or sterile saline by techniques known tothose skilled in the art.

Intravitreal delivery can include single or multiple intravitrealinjections, or via an implantable intravitreal device that releases thecompound in a sustained capacity. Intravitreal delivery can also includedelivery during surgical manipulations as either an adjunct to theintraocular irrigation solution or applied directly to the vitreousduring the surgical procedure.

For systemic administration, plasma concentrations of active compoundsdelivered can vary according to compounds; but are generally1×10⁻⁶–1×10⁻¹ moles/liter, and preferably 1×10⁻⁴–1×10⁻² moles/liter.

The present invention also provides novel formulation compositions ofmatter. The compositions are pharmaceutically acceptable formulationcomprising compounds of Formula I of high purity, and/or in apharmaceutically acceptable carrier. The pharmaceutically acceptablecarrier can be selected by those skilled in the art using conventionalcriteria. The pharmaceutically acceptable carrier include, but are notlimited to, saline and aqueous electrolyte solutions, water polyetherssuch as polyethylene glycol, polyvinyls such as polyvinyl alcohol andpovidone, cellulose derivatives such as methylcellulose andhydroxypropyl methylcellulose, petroleum derivatives such as mineral oiland white petrolatum, animal fats such as lanolin, polymers of acrylicacid such as carboxypolymethylene gel, vegetable fats such as peanut oiland polysaccharides such as dextrans, and glycosaminoglycans such assodium hyaluronate and salts such as sodium chloride and potassiumchloride.

Preferred compounds of the present invention comprise compounds ofFormula I wherein A has a molecular weight of no more than about 1000and is OR₁, SR₁, NR₁R₂, or CR₁R₂R₃ such that R₁, R₂, and R₃ areindependently hydrogen, C₁₋₅₀ alkyl, C₅₋₃₅ cycloalkyl, aryl, arylalkyl,phosphonate, or acylthioalkyl, with or without substituents orheteroatoms; or taken together to form a cycloalkyl or aryl ring, withor without substituents or heteroatoms with the exception of OR₁ and SR₁not being OH or SH; or a natural or non-natural amino acid, peptide,polypeptide, or other oligomer; or natural or non-natural steroid: Morepreferably A is a hydroxylated alkyl group (e.g. glycerol, cholesterol);is an amino acid (e.g. phenylalanine, serine, tyrosine) having 3 to 50carbon atoms; is amino or mono- or disubstituted amino, where thesubstituents are alkyl, cycloalkyl, aralkyl, aryl, substituted aralkyl,or substituted aryl having 3 to 50 carbon atoms and which may alsocontain heteroatoms (e.g. S, N, O,) with 3 to 30 atoms being mostpreferred.

In preferred compounds of the present invention, X₁, X₂, and X₃ ofFormula I are independently oxygen, methylene, monochloromethylene,dichloromethylene, monofluoromethylene, difluoromethylene, or imido.More preferably X₁, X₂, and X₃ are oxygen, dichloromethylene ordifluoromethylene with oxygen being most preferred. In preferredcompounds of the compositions of the present invention, T₁, T₂, W, and Vof Formula I are independently oxygen or sulfur. More preferably T₁ andT₂, are sulfur or oxygen, and W and V oxygen, respectively; with T₁, T₂,W, and V being oxygen being most preferred. In preferred compounds ofthe compositions of the present invention, the sum of m+n+p of Formulais from 1 to 4. More preferably, the sum of m+n+p of Formula is 2 or 3,with 3 being most preferred. In preferred compounds of the presentinvention, M is lithium, sodium or potassium; an alkaline earth metalsalt such as magnesium or calcium; or an ammonium or tetraalkyl ammoniumsalt, i.e., NX₄ ⁺ (wherein X is C₁₋₄). More preferably M is sodium,potassium, or tetraalkyl ammonium; with sodium being most preferred. Inpreferred compounds of the present invention, D is oxygen.

In preferred compounds of the present invention, B is a purine orpyrimidine; with a pyrimidine being most preferred. In preferredcompounds of the present invention, Y is H, OH, or OR, and Z is H, OH,or OR. More preferably both Y and Z are OH. In preferred compounds ofthe present invention, R₄ is H, R₅ is H, R₆ and R₇ together is oxygen,R₈ is mono- or di-substituted amino, R₉ is H or aralkyl, R₁₀ is aralkyl,R₁₁ is alkylamino, R₁₂ is H, R₁₃ is H or halogen, R₁₄ is H, halogen,thioalkyl, or thioaralkyl, R₁₅ is O, S, amino, or substituted amino, R₁₆is H or R₁₅ and R₁₆ are taken together to form a substituted 5-memberedimidazole ring, R₁₇ is H, halogen, alkyl, or substituted alkynyl, andR₁₈ is aralkyloxy. More preferably R₁₃ is H, R₁₄ is H or thioalkyl, R₁₅is O, S, or amino or R₁₅ and R₁₆ taken together to form a substituted5-membered imidazole ring, and R₁₇ is H, halogen, alkyl, or substitutedalkynyl; with R₁₅ is O and R₁₇ is H being most preferred.

A preferred formula for the compound of the present invention is FormulaIa:

wherein the variable groups have the definitions as above.However, A is preferably O-alkyl, O-cycloalkyl, O-aryl, S-alkyl, S-aryl,N-alkyl, N-cycloalkyl, or C-alkyl; more preferably preferably O-alkyl,O-cycloalkyl, or O-aryl,having 3 to 30 carbon atoms;

-   X₁, X₂, and X₃ are preferably oxygen;-   T₁, T₂, W, and V are preferably oxygen;-   preferably the sum of m+n+p is from 1 to 4; more preferably 2 or 3;-   M is preferably H or an alkali metal; more preferably every M is a    sodium or a potassium counter ion;-   D is preferably oxygen;-   B is preferably selected from the group consisting of uracil,    cytosine, thymine, imidazo[1,2-c]pyrimidin-5(6H)-one    {ethenocytosine}, 2-phenyl-imidazo[1,2-c]pyrimidin-5(6H)-one    {phenylethenocytosine}, 5-iodouracil, 5-iodocytosine, 4-thiouracil,    and 5-phenylethynyluracil;-   Y and Z are both preferably OH.

The following compounds, within the scope of the present invention, aredeemed particularly useful:

Structures 1–36 exemplify pyrimidine diphosphates where A=OR₁:

Structures 37–72 exemplify pyrimidine diphosphates where A=CR₁R₂R₃:

Structures 73–130 exemplify pyrimidine triphosphates and tetraphosphateswhere A=OR₁, SR₁, or NR₁R2:

Structures 131–168 exemplify pyrimidine triphosphates andtetraphosphates where A=CR₁R₂R₃:

Structures 169–192 exemplify adenosine triphosphates and tetraphosphateswhere A=OR₁, SR₁,or NR₁R₂:

Structures 193–216 exemplify adenosine triphosphates and tetraphosphateswhere A=CR₁R₂R₃:

More preferred compounds from the structures above (with the abovecompound number in parentheses) include:

2′3′-O-methylenebenzyl β-(cyclohexyl) UDP (5), 2′-phenylcarbamoylβ-benzyl UDP (14), 2′-(phenoxy)formyl β-propyl UDP (15),6-phenyl-furanopyrimidine riboside β-(3-carboxyphenyl)methyl diphosphate(20), 4-thiobenzyl pyrimidine riboside β-benzyl diphosphate (21),2′,3′-dibenzoyl β-propyl UDP (29), 5-(3-methoxyphenyl)ethenocytosine2′-deoxy-3′-phenylcarbamoyl riboside β-propyl diphosphate (33),N⁴-propyl-2′,3′-dibenzoyl β-benzyl CDP (36), 2′3′-O-methylenebenzylβ-(2-methylpropylphosphono) UDP (37), 2′-phenylcarbamoylβ-(2-carboxyethylphosphono) UDP (48), N⁴-(4-fluorophenylcarbamoyl)β-(o-methylbenzylphosphono) CDP (54), 2′,3′-di(phenoxy)formylβ-(pentylphosphono) UDP (61), N⁴-propyl-2′,3′-dibenzoylβ-(2-carboxyethylphosphono) CDP (72), 2′-deoxy γ-benzyl UTP (77),γ-(thiocyclohexyl) UTP (79), 6-(3-methylphenyl)-furanopyrimidineriboside δ-(2-naphthalenemethyl) tetraphosphate (86),2′3′-O-methylenebenzyl γ-propyl UTP (93),5-(3-methylphenyl)ethenocytosine 2′3′-O-methylenebenzyl ribosideδ-propyl tetraphosphate (105), 5-(3-methoxyphenyl)ethenocytidineriboside γ-(2-naphthalenemethyl) triphosphate (111),N⁴-(benzyloxyformyl)-2′-deoxy γ-benzyl CTP (115), N⁴,3′-dibenzoyl-2′-deoxy γ-(2-naphthalmethyl) CTP (123), 5-(3-triphosphate(135), 4-thiopropyl pyrimidine riboside γ-(4-aminocarboxybutylphosphono)triphosphate (138), 2′3′-O-methylenephenethylγ-(3,4-dimethylphenylphosphono) UTP (147), 5-iodo-2′3′-O-methylenebutylγ-(1-naphthalenemethylphosphono) UTP (157), 2′,3′-dibenzoylδ-(4-ethoxyphenylphosphono) uridine tetraphosphate (161),2′3′-O-methylenebenzyl γ-(2-naphthalene) ATP (175),2-thiopropyl-2′3′-O-methylenebenzyl γ-benzyl ATP (180),2-thiomethyl-N⁶-propyl-2′3′-O-methylenebenzyl γ-(2-naphthalene) ATP(183), 2′3′-O-methylenebenzyl γ-anilino ATP (192),2′3′-O-methylenebenzyl γ-(carboxymethylphosphono) ATP (200),2′3′-O-methylenebenzyl δ-(1-naphthalene) adenosine tetraphosphate (201),2-thiopropyl-2′-deoxy-3′-(3-trifluoromethylphenyl)carbamoylγ-(4-methoxyphenylphosphono) ATP (212); with the following compoundsbeing most preferred; 2′3′-O-methylenebenzyl β-(cyclohexyl) UDP (5),5-(3-methoxyphenyl)ethenocytosine 2′-deoxy-3′-phenylcarbamoyl ribosideβ-propyl diphosphate (33), 2′3′-O-methylenebenzylβ-(2-methylpropylphosphono) UDP (37), 2′,3′-di(phenoxy)formylβ-(pentylphosphono) UDP (61), 2′3′-O-methylenebenzyl γ-(propyl) UTP(93), 5-(3-methylphenyl)ethenocytosine 2′3′-O-methylenebenzyl ribosideδ-propyl tetraphosphate (105), 5-(3-trifluoromethylphenyl)ethenocytidineγ-(1-naphthalenemethylphosphono) triphosphate (135), 2′,3′-dibenzoylδ-(4-ethoxyphenylphosphono) uridine tetraphosphate (161),2-thiopropyl-2′3′-O-methylenebenzyl γ-benzyl ATP (180), and ),2′3′-O-methylenebenzyl δ-(1-naphthalene) adenosine tetraphosphate (201).

The invention is illustrated further by the following examples that arenot to be construed as limiting the invention in scope to the specificprocedures described in them.

EXAMPLES Example 1 γ-(n-propyl)-uridine 5′-triphosphate

Uridine 5′-triphosphate, ditributylammonium salt ( 106 mg, 0.124 mmol)dissolved in dry N,N dimethylformamide (400 uL) was treated withN,N′-dicyclohexlycarbodiimide (33.4 mg, 0.162 mmol) for one hour at roomtemperature. After verifying that there was complete conversion to thecyclical trimetaphosphate by ³¹P NMR, tributylamine (88 μL, 0.373 mmol)and excess n-propanol (1 mL) were added and the reaction mixture heatedto 65° C. for 2.5 days. HPLC (AX300, gradient from 75% water/25%acetonitrile to 75% 0.5 M KH2PO4 over 20 min, 1 mL/min, monitor at 260nm) showed >90% conversion to product, so the solvents were removed on arotary evaporator. The product was purified by semi-preparative HPLC(AX300, gradient from 75% water/25% acetonitrile to 75% 1 M ammoniumacetate/25% acetonitrile over 20 min, 2 mL/min, monitor at 260 nm)yielding 19.5 mg (28%) of the title product.

¹H NMR (D₂O, 300 MHz): δ 7.78 (d, 1H), 5.79 (m, 2H), 4.19 (M, 2H), 4.04(m, 3H), 3.73 (q, 2H), 1.46 (m, 2H), 0.72 (t, 3H). ³¹p NMR (D₂O, 121.47MHz): δ −9.60 (d, 1P), −10.34 (d, 1P), −21.98 (t, 1P).

Example 2 γ-(2-propyl)-uridine 5′-triphosphate

The title product was obtained from the reaction between uridine5′-triphosphate and 2-propyl alcohol, according to the method ofexample 1. Yield=14%.

¹H NMR (D₂O, 300 MHz): δ 7.80 (d, 1H), 5.81 (m, 2H), 4.23 (M, 1H), 4.11(m, 2H), 4.07 (m, 3H), 1.10 (d, 6H). ³¹P NMR(D₂O, 121.47 MHz): δ −9.60(d, 1P), −10.34 (d, 1P), −21.98 (t, 1P).

Example 3 γ-(n-butyl)-uridine 5′-triphosphate

The title product was obtained from the reaction between uridine5′-triphosphate and n-butyl alcohol, according to the method ofexample 1. Yield=39%.

¹H NMR (D₂O, 300 MHz): δ 7.82 (d, 1H), 5.83 (m, 2H), 4.22 (M, 1H), 4.11(m, 3H), 3.80 (q, 2H), 1.46 (t, 2H), 1.21 (q, 2H), 0.72 (t, 3H). ³¹P NMR(D₂O, 121.47 MHz): δ −9.44 (d, 1P),−10.18 (d, 1P), −21.82 (t, 1P).

Example 4 γ-(n-hexyl)-uridine 5′-triphosphate

The title product was obtained from the reaction between uridine5′-triphosphate and n-hexyl alcohol, according to the method ofexample 1. Yield=12%.

¹H NMR (D₂O, 300 MHz): δ 7.80 (d, 1H), 5.81 (d, 2H), 4.20 (M, 2H), 4.11(m, 3H), 3.78 (q, 2H), 1.41 (t, 2H), 1.09 (q, 6H), 0.68 (t, 3H). ³¹P NMR(D₂O, 121.47 MHz): δ −9.58 (d, 1P), −10.22 (d, 1P), −21.68 (t, 1P).

Example 5 γ-(farnesyl)-uridine 5′-triphosphate

The title product was obtained from the reaction between uridine5′-triphosphate and farnesol (as a mixture of isomers), according to themethod of example 1. Yield=8%, as an unstable solid.

¹H NMR (D₂O, 300 MHz): δ 7.78 (d, 1H), 5.87 (d, 2H), 5.22 (m, 1H), 4.98(m, 2H), 4.23 (m, 2H), 4.18 (m, 3H), 4.01 (m, 2H), 1.81 (t, 8H), 1.24(d, 12H). ³¹P NMR (D₂O, 121.47 MHz): δ −9.69 (d, 1P), −10.18 (d, 1P),−21.88 (t, 1P).

Example 6 γ-(cholesteryl)-uridine 5′-triphosphate

The title product is obtained from the reaction between uridine5′-triphosphate and cholesterol, according to the general method ofexample 1. If necessary, this process can be enhanced by the addition ofcatalysts such as pyridine, 4-dimethylaminopyridine,diazabicycloundecene (DBU) and the like.

As demonstrated in the preceding examples 1–6, different nucleophilescan be used to open the cyclical trimetaphosphate, giving derivativeswith unique substituents on the γ phosphate. Thus, for example, theactivated triphosphate can be reacted with 2-naphthylmethyl alcohol togive structure 91, or with cyclohexylmethyl alcohol to give 78.Alternately, nitrogen nucleophiles (giving products such as structures81 and 82), or sulfur nucleophiles (giving products such as 79 and 89)can be used. Yet another choice would be the use of phosphatenucleophiles, giving δ-substituted tetraphosphate derivatives fallingwithin the scope of the invention. Thus, for example, treatment of thecyclical trimetaphosphate with 2-naphthylmethyl phosphate would yieldstructure 76, while benzyl phosphate would yield 85. Yet another choicewould be the use of phosphonic acid derivatives as nucleophiles, againgiving δ-substituted tetraphosphate derivatives falling within the scopeof the invention (for example, structures 131, 132, and 133).

Finally, the above Examples are illustrative and more elaboratemolecules can be used to generate other compounds within the scope ofthe present invention, bearing novel substituents on the sugar and/orthe base, by use of the appropriate reagents.

Example 7 2′,3′-((benzyl)methylenedioxy)-γ-(n-propyl)-uridine5′-triphosphate

Uridine 5′-triphosphate, trisodium salt (1.0 g, 1.82 mmol) was dissolvedin 98% formic acid (5 mL) and phenylacetaldehyde, dimethyl acetal (602uL, 3.64 mmol) added. The reaction was stirred overnight at ambienttemperature, at which point TLC (silica gel, 50% isopropanol/50%ammonium hydroxide) and HPLC (C18) showed good conversion to a lesspolar product. The formic acid was removed on a rotary evaporator, andthe residue partitioned between 1 M sodium bicarbonate (15 mL) and ethylacetate (25 mL). The layers were separated and the aqueous was washedwith a further portion of ethyl acetate (25 mL). The aqueous layer wasstripped and the residue lyophilized overnight. The crude product wasdissolved in water (5 mL) and the components separated by preparativeHPLC (Waters Novapak C18, 6 um, 25×100 mm, gradient from 0.1 M ammoniumacetate to methanol over 30 minutes, 30 mL/min, monitor at 260 nm). Theyield of the acetal was 352 mg (30%).

¹H NMR (D₂O, 300 MHz): δ 7.62 (d, 1H), 7.22 (m, 5H), 5.73 (d, 1H), 5.40(d, 1H), 5.32 (t, 1H), 4.69 (m, 2H), 4.33 (m, 1H), 4.00 (m, 2H), 3.01(d, 2H). ³¹P NMR (D₂O, 121.47 MHz): δ −7.47 (d, 1P), −10.54 (d, 1P),−21.46 (t, 1P).

The title compound is obtained by further manipulation according to themethod of example 1.

Example 8 2′,3′-((benzyl)methylenedioxy)-uridine 5′-monophosphate

Uridine 5′-monophosphate, disodium salt (1.0 g, 2.72 mmol) was dissolvedin 98% formic acid (7.5 mL) and phenylacetaldehyde, dimethyl acetal (900uL, 5.44 mmol) added. The reaction was stirred for 2 days at 30° C.,after which the formic acid was removed and the residue partitionedbetween 1 M sodium bicarbonate (20 mL) and ethyl acetate (20 mL). Thelayers were separated and the aqueous was extracted once more with ethylacetate (20 mL). The aqueous layer was concentrated to 8 mL and theproduct separated using preparative HPLC, as described in example 7.Yield=241 mg (19%).

The product so obtained is converted to the monotributylammonium salt bydirect treatment with an excess of tributylamine in aqueous methanol,after which it is dried by repeated evaporation with dry N,Ndimethylformamide. This is treated with 1, 1′-carbonyldiimidazole toactivate the monophosphate as the corresponding imidazolide, which iscoupled with a variety of phosphate-containing nucleophiles, such ascyclohexylphosphate (giving structure 5), n-propylphosphate (giving 2),benzyl phosphate (giving 10), isobutylphosphonic acid (giving 37),1-naphthylmethylphosphonic acid (giving 41), and n-hexylphosphonic acid(giving 45).

By this general method, a variety of diphosphates bearing substituentson the β phosphate falling within the scope of this invention can beproduced. These diphosphates can be further modified with other groupson the sugar and/or the base, as previously described for tri- andtetraphosphates.

Example 10 Uridine 5′-monophosphate (2-fluoro-4-nitrobenzyl)ester

A solution of 2′,3′- O-isopropylidene-uridine 5′-monophosphoric acid(0.2 mmol) and 2-fluoro-4-nitrobenzyl alcohol (0.4 mmol) in pyridine (5mL) is treated with triphenylphosphine (6 mmol) anddiethylazodicarboxylate (4 mmol) 7 hours at 28° C. The solvents areremoved in vacuo and the residue chromatographed on silica gel withmethanol-chloroform as eluent, to isolate 2′,3′-O-isopropylidene-uridine5′-monophosphate (2-fluoro-4-nitrobenzyl)ester. This compound is treatedwith methanolic HCl 20 minutes at 28° C., and the solvent removed invacuo to afford the title compound.

Example 11 Parallel Synthesis of Nucleotide Aryl Phosphodiesters

An 8×8 array of 2′,3′-O-isopropylidene-uridine and2′,3′-O-isopropylidene—6-N-benzoy-adenosine (0.1 mmol) in a reactionblock are dissolved in dichloromethane (3 mL/well) and treated with2-cyanoethyl-N,N′-diisopropyl chlorophosphoramidite (0.2 mmol) andtriethylamine (0.4 mmol) per well 20 minutes at 25° C. on a shaker. Thesolvent is evaporated under a stream of nitrogen and the block dried ina vacuum oven overnight. To each of the uridine-containing wells isadded 0.2 mmol of a different alcohol selected from the group of TableA, as a solution in dichloromethane. The process is repeated for theadenosine-containing wells. Tetrazole (0.3 mmol) is added and the arrayis shaken 10 minutes at 25° C. The reaction block is next immersed in aCO₂-acetonitrile bath and mCPBA (0.5 mmol in 1 mL THF/well) added. Theblock is shaken and allowed to come to room temperature. An additional 2mL of dichloromethane per well is added and each well extracted with 5%sodium thiosulfite (2×1 mL) followed by 10% sodium bicarbonate (1×1 mL)and brine (1×1 mL). The organic phase is made 50% in trifluoroaceticacid and shaken 60 minutes at 25° C. The organic phase is again driedunder nitrogen and treated with aqueous ammonia in THF overnight. It isthen evaporated under a stream of nitrogen, and the residue extractedwith ether (3×1 mL). Each product is solubilized in alcohol and appliedto the top of individual C18 extraction columns. Nitrogen is passedthrough the column to evaporate the alcohol and the column is elutedstepwise with water, 10% methanol-water, 25% methanol-water, and 50%methanol-water to afford the desired products.

TABLE A Coupling Partners for 2′, 3′-O-isopropylidene-uridine5′-phosphoramidate and 2′, 3′-O-isopropylidene-6-N-benzoyl-adenosine5′-phosphoramidate.

It should be apparent that given the guidance, illustrations andexamples provided herein, various alternate embodiments, modificationsor manipulations of the present invention would be suggested to askilled artisan and these are included within the spirit and purview ofthis application and scope of the expanded claims.

1. A compound of Formula I, or a pharmaceutically acceptable saltthereof:

wherein A is a covalently bound substituent having a maximum molecularweight of 1000 and is OR₁ or SR₁, wherein R₁ is cycloalkyl withoutsubstituents, aryl, arylalkyl, phosphonate, or acylthioalkyl with orwithout substituents or heteroatoms; X₁, X₂, and X₃ are independentlyoxygen, methylene, monochloromethylene, dichloromethylene,monofluoromethylene, difluoromethylene, or imido; T₁, T₂, W, and V areindependently oxygen or sulfur; m=0, 1, or 2; n=0 or 1; p=0, 1, or 2;where the sum of m+n+p is from 0 to 5; M=H or apharmaceutically-acceptable inorganic or organic counter ion; D=O orCH₂; B is a purine or a pyrimidine residue according to general FormulaeIV and V which is linked to the 1′ position of the furanose orcarbocycle via the 9- or 1- position of the base, respectively; Y=H, OH,or OR₄; Z=H, OH, or OR₅; with the proviso that Y and Z are both not H;R₄ and R₅ are residues which are linked directly to the 2′ and/or 3′oxygens of the furanose or carbocycle via a carbon atom according toFormula II, or linked directly to the two 2′ and 3′ oxygens of thefuranose or carbocycle via a common carbon atom according to FormulaIII;

wherein: O is the corresponding 2′ and/or 3′ oxygen of the furanose orcarbocycle; C is the carbon atom; R₆, R₇, and R₈ are H, an alkyl,cycloalkyl, aralkyl, aryl, substituted aralkyl, or substituted aryl,such that the moiety defined according to Formula II is an ether; or R₆and R₇ are H, an alkyl, cycloalkyl, aralkyl, aryl, substituted aralkyl,or substituted aryl, and R₈ is alkoxy, cycloalkoxy, aralkyloxy, aryloxy,substituted aralkyloxy, or substituted aryloxy such that the moietydefined according to formula II is an acyclic acetal or ketal; or R₆ andR₇ are taken together as oxygen or sulfur doubly bonded to C, and R₈ isalkyl, cycloalkyl, aralkyl, aryl, substituted aralkyl, or substitutedaryl, such that the moiety defined according to Formula II is an esteror thioester; or R₆ and R₇ are taken together as oxygen or sulfur doublybonded to C, and R₈ is amino or mono- or disubstituted amino, where thesubstituents are alkyl, cycloalkyl, aralkyl, aryl, substituted aralkyl,or substituted aryl, such that the moiety according to Formula II is acarbamate or thiocarbamate; or R₆ and R₇ are taken together as oxygen orsulfur doubly bonded to C, and R₈ is alkoxy, cycloalkoxy, aralkyloxy,aryloxy, substituted aralkyloxy, or substituted aryloxy, such that themoiety according to Formula H is a carbonate or thiocarbonate; or R₈ isnot present and R₆ and R₇ are taken together as oxygen or sulfur doublybonded to C and both the 2′ and 3′ oxygens of the furanose are directlybound to C to form a cyclical carbonate or thiocarbonate;

wherein: O is the 2′ and 3′ oxygens of the furanose or carbocycle; andthe 2′ and 3′ oxygens of the furanose or carbocycle are linked by acommon carbon atom to form a cyclical acetal, cyclical ketal, orcyclical orthoester; for cyclical acetals and ketals, R₉ and R₁₀ areindependently hydrogen, alkyl, cycloalkyl, aralkyl, aryl, substitutedaralkyl, substituted aryl, or can be joined together to form ahomocyclic or heterocyclic ring composed of 3 to 8 atoms; for cyclicalorthoesters, R₉ is hydrogen, alkyl, cycloalkyl, aralkyl, aryl,substituted aralkyl, or substituted aryl, R₁₀ is alkyloxy,cycloalkyloxy, aralkyloxy, aryloxy, substituted aralkyloxy, orsubstituted aryloxy;

wherein: R₁₁ and R₁₅ are hydroxy, oxo, amino, mercapto, alkylthio,alkyloxy, aryloxy, alkylamino, cycloalkylamino, aralkylamino, arylamino,diaralkylamino, diarylamino, or dialkylamino, where the alkyl groups areoptionally linked to form a heterocycle; or R₁₁ and R₁₅ are acylamino;or when R₁₁ in a purine or R₁₅ in a pyrimidine has as its first atomnitrogen, R₁₁ and R₁₂ or R₁₅ and R₁₆ are taken together to form a5-membered fused imidazole ring optionally substituted on the ethenoring with alkyl, cycloalkyl, aralkyl, or aryl moieties; when R₁₅ in apyrimidine has as its first atom oxygen, R₁₅ and R₁₇ are taken togetherto form a 5-membered dihydrofuran ring, optionally substituted on thedihydrofuran ring with alkyl, cycloalkyl, aralkyl, or aryl moieties; Jis carbon or nitrogen, with the provision that when nitrogen, R₁₃ is notpresent; R₁₂ is hydrogen, O or is absent; R₁₆ is hydrogen, or acyl; R₁₃is hydrogen, alkyl, bromo, azido, alkylamino, arylamino or aralkylamino,alkoxy, aryloxy or aralkyloxy, alkylthio, arythio or aralkylthio, orω-E(C₁₋₆alkyl)G-, wherein E and G are independently amino, mercapto,hydroxy or carboxyl; R₁₄ is hydrogen, chlorine, amino, monosubstitutedamino, disubstituted amino, alkylthio, arylthio, or aralkylthio, wherethe substituent on sulfur contains up to a maximum of 20 carbon atoms,with or without unsaturation; and R₁₇ is hydrogen, methyl, alkyl, halo,alkyl, alkenyl, substituted alkenyl, alkynyl, or substituted alkynyl. 2.The compound according to claim 1, wherein: A is OR₁ or SR₁, wherein R₁is cycloalkyl without substituents, aryl, arylalkyl, phosphonate, oracylthioalkyl with or without substituents or heteroatoms; X₁, X₂, andX₃ are each oxygen; T₁, T₂, W, and V are each oxygen; D=O.
 3. Thecompound according to claim 1, wherein Formula I is a compound ofFormula Ia:

wherein the variable groups have the definitions as described inclaim
 1. 4. A pharmaceutical composition comprising a compound ofFormula I of claim 1 in a pharmacologically acceptable carrier.
 5. Acompound selected from the group consisting of: 2′3′-O-methylenebenzylβ-(cyclohexyl) UDP, 2′-phenylcarbamoyl β-benzyl UDP, 2′-(phenoxy)formylβ-propyl UDP, 6-phenyl-furanopyrimidine ribosideβ-(3-carboxyphenyl)methyl diphosphate, 4-thiobenzyl pyrimidine ribosideβ-benzyl diphosphate, 2′,3′-dibenzoyl β-propyl UDP,5-(3-methoxyphenyl)ethenocytosine 2′-deoxy-3′-phenylcarbamoyl ribosideβ-propyl diphosphate, N⁴-propyl-2′,3′-dibenzoyl β-benzyl CDP, 2′-deoxyγ-benzyl UTP, γ-(thiocyclohexyl) UTP,6-(3-methylphenyl)-furanopyrimidine riboside δ-(2-naphthalenemethyl)tetraphosphate, 2′3′-O-methylenebenzyl γ-propyl UTP,5-(3-methylphenyl)ethenocytosine 2′3′-O-methylenebenzyl ribosideδ-propyl tetraphosphate, 5-(3-methoxyphenyl)ethenocytidine ribosideγ-(2-naphthalenemethyl) triphosphate, N⁴-(benzyloxyformyl)-2′-deoxyγ-benzyl CTP, N⁴,3′-dibenzoyl-2′-deoxy γ-(2-naphthalmethyl) CTP,2′3′-O-methylenebenzyl γ-(2-naphthalene) ATP,2-thiopropyl-2′3′-O-methylenebenzyl γ-benzyl ATP, and2-thiomethyl-N⁶-propyl-2′3′-O-methylenebenzyl γ-(2-naphthalene) ATP. 6.The compound according to claim 5, wherein the compound is selected fromthe group consisting of: 2′3′-O-methylenebenzyl β-(cyclohexyl) UDP,5-(3-methoxyphenyl)ethenocytosine 2′-deoxy-3′-phenylcarbamoyl ribosideβ-propyl diphosphate, 2′3′-O-methylenebenzyl γ-(propyl) UTP,5-(3-methylphenyl)ethenocytosine 2′3′-O-methylenebenzyl ribosideδ-propyl tetraphosphate, and 2-thiopropyl-2′3′-O-methylenebenzylγ-benzyl ATP.
 7. The pharmaceutical composition according to claim 4,wherein the compound is in a formulation selected from the groupconsisting of: aqueous solution, liquid/liquid suspension, gel,gel-like, and solid formulations.